Journal: bioRxiv

Article Title: Direct effects on endothelial cells are essential for perivascular cell (pericyte)-dependent amplification of orthoflavivirus NS1-mediated microvascular leakage

doi: 10.64898/2026.01.29.702573

Figure Lengend Snippet: (A) Representative images of HUVECs forming vessel-like structures in 3D Geltrex® matrix in the presence of HSCs, with or without 1 µg/mL NS1 treatment. Scale bars, 100 µm. (B) Angiogenesis assay quantification method. “Junctions” connect vessel-like structures; “branches” connect to junctions at one end; “segments” connect at both ends. “Total branching length” includes all branches; counts reflect the total number of segments, branches and junctions within field of view. (C-F) Quantification of vessel-like structures in pericyte-endothelial cell cocultures following NS1 treatment, normalised to untreated HUVECs (dashed line). Data points represent technical replicates within an experiment with different symbol shapes corresponding to different experiments (N=4, n=4). Bars represent the mean, and error bars represent SEM. Indicated significance is for treatment versus untreated cocultures (Untreated). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Plasmids encoding Streptavidin-tagged NS1 from AHFV (pKM74; viral reference sequence NCBI accession JF416957.1 ) or KFDV (pKM79 accession JF416958.1 ) were transfected into CHO DG44 cells using FreeStyle MAX reagent (ThermoFisher), followed by selection using 0.3 μg/mL geneticin (G418 sulphate, ThermoFisher).

Techniques: Angiogenesis Assay

Journal: bioRxiv

Article Title: Direct effects on endothelial cells are essential for perivascular cell (pericyte)-dependent amplification of orthoflavivirus NS1-mediated microvascular leakage

doi: 10.64898/2026.01.29.702573

Figure Lengend Snippet: (A-B) ECIS measurement of permeability of monocultures of HUVECs (A) or LSECs (B) treated with preconditioned media from HSCs treated with 1 µg/mL NS1 or 100 ng/mL TNF-α for 24 h; “Untreated” refers to HUVECs or LSECs treated with HSC media only and the “EC only” refers to untreated HUVECs or LSECs without the addition of HSC media. (C, D) Dextran permeability assay measuring HUVEC (C) or LSEC (D) permeability in coculture with HSCs 24 h post-treatment with 1 µg/mL NS1, 100 ng/mL TNF-α (positive control), or eluate (negative) control. Data points represent technical replicates within an experiment with different symbol shapes corresponding to different experiments (N=3, n=3). Bars represent the mean, and error bars represent SEM. (E, F) TEER measurement of semi-contact cocultures of HUVECs (A) or LSECs (B) cultured with HSCs and treated with 1 µg/mL NS1, 100 ng/mL TNF-α (positive control), or eluate (negative) control. Permeability measurements were normalised to each sample pre-treatment for ECIS data (A, B) or the untreated endothelial cell only control (EC only; dashed line) for EVOM data (E, F). In the time course experiments (A, B, E, F), each data point represents the mean ± SEM. In all experiments indicated significance is compared to untreated cocultures; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Plasmids encoding Streptavidin-tagged NS1 from AHFV (pKM74; viral reference sequence NCBI accession JF416957.1 ) or KFDV (pKM79 accession JF416958.1 ) were transfected into CHO DG44 cells using FreeStyle MAX reagent (ThermoFisher), followed by selection using 0.3 μg/mL geneticin (G418 sulphate, ThermoFisher).

Techniques: Permeability, FITC-Dextran Permeability Assay, Positive Control, Negative Control, Cell Culture, Control

Journal: bioRxiv

Article Title: Direct effects on endothelial cells are essential for perivascular cell (pericyte)-dependent amplification of orthoflavivirus NS1-mediated microvascular leakage

doi: 10.64898/2026.01.29.702573

Figure Lengend Snippet: Endothelial barrier integrity measurements for HUVEC (A, B) or LSEC (C) monocultures following treatment with 1 µg/mL wild-type DENV-2, AHFV or KFDV NS1, or the nonfunctional DENV-2 NS207Q mutant (negative control), or 100 ng/mL TNF-α (positive control), or eluate (negative) control as indicated. Permeability measurements were normalised to the untreated endothelial cell only control (Untreated) for EVOM TEER data (A), or to each sample before treatment for ECIS data (B, C). Each data point represents the mean ± SEM. Indicated significance is for treatment versus untreated EC only control (Untreated). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Plasmids encoding Streptavidin-tagged NS1 from AHFV (pKM74; viral reference sequence NCBI accession JF416957.1 ) or KFDV (pKM79 accession JF416958.1 ) were transfected into CHO DG44 cells using FreeStyle MAX reagent (ThermoFisher), followed by selection using 0.3 μg/mL geneticin (G418 sulphate, ThermoFisher).

Techniques: Mutagenesis, Negative Control, Positive Control, Permeability, Control